is n -acetyl cysteine protective against monocrotaline-induced toxicity?

2013

http://informahealthcare.com/txrISSN: 1556-9543 (print), 1556-9551 (electronic)

Toxin Rev, 2013; 32(3): 47–54! 2013 Informa Healthcare USA, Inc. DOI: 10.3109/15569543.2013.809547

REVIEW ARTICLE

Is N-acetyl cysteine protective against monocrotaline-induced toxicity?

Serife Karagoz1, Sinem Ilgin1, Ozlem Atli1, Basak Ozlem Perk1, Dilek Burukoglu2, Bulent Ergun1, and Basar Sirmagul3

1Department of Pharmaceutical Toxicology, Faculty of Pharmacy, Anadolu University, Eskisehir, Turkey, 2Department of Histology, and3Department of Pharmacology, Faculty of Medicine, Osmangazi University, Eskisehir, Turkey

Abstract

Monocrotaline (MCT) is a pyrrolizidine alkaloid which induces cardio-pulmonary toxicity andhepatotoxicity in animals and humans. MCT is frequently ingested because of food graincontamination accidentally or in the form of herbal medicine preparations. The aim of this studywas to observe the protective effect of N-acetyl cysteine (NAC) on MCT-induced pulmonarytoxicity and hepatotoxicity. According to our results (right ventricular pressures [RVPs], ratios ofright ventricle (RV)/heart weight (HW), plasma AST levels, liver glutathione levels, liver MDA levelsand liver histopathological examinations of groups), protective effects were observed with NACtreatment in both MCT-induced pulmonary toxicity and hepatotoxicity.

Keywords

Glutathione, hepatotoxicity, monocrotaline,N-acetyl cysteine, pulmonary toxicity

History

Received 16 April 2013Revised 24 May 2013Accepted 25 May 2013Published online 19 July 2013

Introduction

Monocrotaline (MCT) is a pyrrolizidine alkaloid present in the

plants of the Crotalaria species that causes pulmonary toxicity

and hepatotoxicity in animals and humans. MCT is frequently

ingested accidentally because of food grain contamination or in

the form of herbal medicine preparations (Copple et al., 2003).

Numerous cases of human and animal poisoning by pyrroli-

zidine alkaloids were reported around the world. It was

suggested that the consumption of herbal medicines containing

pyrrolizidine alkaloids might contribute to the high incidence

of chronic liver disease and primary liver cancer in Asia and

Africa (Wiedenfield & Edgar, 2011; WHO, 1988). The largest

MCT-related human poisonings occurred in South Asia, two

from central India and one from northwest Afghanistan. The

incidence rate was 1.1% and the case fatality rate was 50% in

these outbreaks (Roeder & Wiedenfeld, 2011). Pyrrolizidine

alkaloids, for example, MCT, show either no or low acute

toxicity, but they can undergo a metabolic toxication process

leading to alkylating agents. This process takes place in human

or animal liver and this organ is therefore the first target organ

for the intoxication (Chen et al., 2009). Toxic metabolites

damage the endothelium of central veins, causing cell prolif-

eration and veno-occlusive disease; metabolites may escape

via the blood stream and induce damage in other organs,

especially lungs. Certain pyrrolizidine alkaloids, for example,

MCT, produce veno-occlusive disease of liver as well as a

sequence of changes in the lungs and heart that result in

pulmonary hypertension and right ventricular hypertrophy

(Klaassen, 2001).

MCT induces toxic effects on the tissues of liver–lung

circulation after being activated by cytochrome P450 enzyme

system in liver and transformed into the reactive form, MCT

pyrrols (Campian et al., 2006; Lame et al., 2000). Pathway

of hepatic glutathione (GSH) conjugation has an important

role in the detoxification of reactive metabolite of MCT which

is occurred as a result of oxidation through cytochrome

oxidase system. Hepatic GSH stores are considerably import-

ant for cardiopulmonary and hepatic toxicity of MCT (Deleve

et al., 1996).

N-Acetyl cysteine (NAC) is an agent which is a

GSH precursor and is used as an antidote in acetaminophen

(AAP) intoxication intravenously and orally because of its

hepatoprotective effect. Also, the protective effects of NAC

were shown in different types of intoxications related to

oxidative stress (Kelly, 1998; Tran et al., 2001; Yousef et al.,

2010). The hepatoprotective effect of NAC was also deter-

mined in tetrachloromethane and azotiopurin-induced hepato-

toxicity models in rats (Raza et al., 2003; Ulicna et al., 2003).

In this study, it was aimed to determine the protective

activity of NAC on hepatotoxicity and pulmonary toxicity

which were induced by MCT in rats. Induction of pulmonary

toxicity was evaluated by the measurement of pulmonary

artery pressure and RV hypertrophy in rats. Hepatotoxicity

was evaluated by serum alanine aminotransferase (ALT),

aspartate aminotransferase (AST), gamma-glutamyl transpep-

tidase (GGT) and alkaline phosphatase (AF) levels in

rats. In addition, samples of liver tissues were investigated

histopathologically. Also, GSH and malondialdehyde (MDA)

levels were determined in tissue homogenates of liver.

Materials and methods

Materials

The chemicals and drugs used were obtained from the

following sources: ketamine (Ketalar�; Phizer, Turkey), MCT

(Sigma, MO), NAC (Asist�; Husnu Arsan, Turkey) and

Address for correspondence: Dr Sinem Ilgin, Department ofPharmaceutical Toxicology, Faculty of Pharmacy, Anadolu University,Eskisehir 26470, Turkey. E-mail: [email protected]

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xylazine (Sigma, MO). For the measurements of GSH levels,

ELISA kits from Cayman Chemical Company was used

according to the manufacturer’s instructions in liver hom-

ogenates. For the measurements of MDA levels, ELISA kits

from Oxis International Inc. was used. Blood AST, ALT, AP

and GGT levels were determined by colorimetric kits from

Biolabo S.A. (Chatel-St-Denis, Switzerland).

Animals

Male Sprague–Dawley rats weighing 250–300 g were

obtained from our own animal facility. Rats were housed

under controlled temperature (22 �C) and lighting (12/12-h

light dark cycle) with free access to food and water. Animal

care and research protocols were based on the principles and

guidelines adapted from the Guide for the Care and Use of

Laboratory Animals (NIH publication No: 85-23, revised

in 1985) and approved by the Local Ethics Committee of

Anadolu University, Eskisehir.

The animals were randomly assigned to different con-

trol and treatment groups (10 animals in each group).

Experimental groups were consisted of: (1) Control: rats

were given a subcutaneous injection of vehicle (1 N HCl,

pH adjusted to 7.4 with 1 N NaOH, 2 mlkg�1). After 2 h,

2 mlkg�1 saline was administered intraperitoneally for 21 d.

(2) MCT: rats were given an intraperitoneal injection of MCT

(60 mgkg�1). After 3 weeks, hemodynamic studies were

performed. (3) NAC: tats were given an intraperitoneal

injection of 60 mgkg�1 MCT. After 2 h, NAC (100 mgkg�1;

2 mlkg�1) was administered intraperitoneally. NAC adminis-

tration was continued intraperitoneally for 21 d.

MCT was dissolved 1 N HCl, and pH was adjusted to 7.4

with 1 N NaOH. MCT (60 mgkg�1) or its vehicle was admin-

istered to rats as a single subcutaneous injection. The MCT

dose was determined according to the previous studies. In

previous studies, pulmonary toxicity was found to be

developed after 21 d by the administration of a single dose

of 60 mgkg�1 MCT (Kang et al., 2003; Liu et al.,

2007; Nagaoka et al., 2005). NAC was dissolved in saline.

NAC dose was determined according to the previous

studies which were performed to test the protective effect

of NAC against hepatotoxicity models (Raza et al., 2003;

Tran et al., 2001).

Measurement of RVP

Rats were anaesthetized with 60 mgkg�1 ketamine and

5 mgkg�1 xylazine. RVP was accessed via a blunt dissection

in the right third and fourth intercostal space, and a 23-gauge

needle placed on the tip of a polyethylene 50 (PE 50) catheter

was inserted into the RV, after which direct RVP recordings

were obtained by Biopac MP150 (Biopac Systems, Inc., CA)

Data Acquisition.

Measurement of right ventricular hypertrophy

The heart was dissected and weighed after excess blood was

removed. The right ventricular wall was separated from

the left ventricle and septum to determine the wet weight.

The ratio of RV to total HW (RV/HW) was calculated to

determine the index of right ventricular hypertrophy.

Biochemical measurements

Blood was collected by cardiac puncture after recording RVP.

The blood samples were centrifuged at 2000� g for 15 min at

4 �C after they were rested at room temperature for 30 min.

Then, the upper layer of serum was pipetted off. AST, ALT,

GGT and AP levels were measured spectrophotometrically in

serum by in vitro diagnostic kits.

Liver tissues were excised from animals. The liver samples

were stored at �80 �C until they were subjected to biochem-

ical analysis. The liver tissues were used for the determination

of GSH and MDA levels in groups.

GSH assay

The tissues were washed with phosphate-buffered saline

(PBS) solution, pH 7.4. They were diluted in the ratio of 1:20

(w:v) with cold buffer (i.e. 50 mM 2-(N-morpholino)

ethanesulfonic acid (MES), pH 6–7, containing 1 mM

EDTA) and were homogenized. The homogenates were

centrifuged at 10000� g for 15 min at 4 �C. The supernatants

were removed and were deproteinated. Then, the samples

were used for total GSH assay. GSH assay was performed

using ELISA kit (Cayman Chemical Company, MI) according

to the manufacturer’s instructions.

MDA assay

The tissues were washed with a PBS solution, pH 7.4. They

were diluted in the ratio of 1:20 (w:v) with cold

buffer (i.e. 0.25 M sodium phosphate buffer, pH 7.4, containing

0.05 M sucrose) and were homogenized. The homogenates

were centrifuged at 10000� g for 15 min at 4 �C. The

supernatants were removed. Then, the samples were used for

assay of MDA. The MDA assay was performed using ELISA

kit (Oxis International, Inc., CA) according to the manufac-

turer’s instructions.

Light microscopic analysis

The liver samples were fixed in a 10% buffered formalin

solution for 48 h and embedded in paraffin. Then, 5 mm thick

slices were stained with hematoxylin and eosin and examined

by light microscopy. All sections were observed under an

Olympus BH-2 (Olympus Corp., Tokyo, Japan) microscope.

Statistical analysis

RVP, RV/HW, AST, ALT, GGT, AP, GSH and MDA ratios

were expressed as mean� standard deviation. Statistical

analyses were performed with one-way ANOVA followed

by Tukey’s HSD test with the GraphPad Prism version 5.0

software (Graphpad Software, Inc., CA). A p value of50.05

was considered statistically significant.

Results

Assessment of RVP

RVP was significantly increased in the MCT group (26.95�1.97 mmHg) when the values were compared with those of

the control group (12.734� 1.47 mmHg). However, in NAC

group, RVP and consequently pulmonary artery pressure were

significantly decreased (18.49� 1.89 mmHg) in comparison

48 S. Karagoz et al. Toxin Rev, 2013; 32(3): 47–54

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with MCT group. Also, significant difference was observed

between control and NAC groups regarding RVP (Figure 1).

Assessment of RV hypertrophy

It was observed that the mass of the RV was significantly

increased in the MCT group when compared with the control

group. The MCT-induced RV hypertrophy was significantly

decreased in NAC group, also the mass of the RV was similar

with the control group (Figure 2).

Serum AST, ALT, AP and GGT levels

Although plasma AST level was significantly increased in

pulmonary hypertension, ALT, GGT and AP levels were not

different in MCT groups (p50.05) when compared with the

levels of the control group (Figure 3). On the other hand,

NAC treatment significantly decreased AST levels in serum

and had no effect on ALT, GGT and AP levels (AST level

234.37� 42.52mM in control and 333.18� 51.02 mM in

MCT, 235.81� 61.66 in NAC groups; p50.05). ALT, GGT

and AP levels were presented in Table 1.

Liver GSH levels

In liver tissue, GSH levels of MCT group were observed to

decrease when they were compared with the control group,

however these values were not significant statistically. On the

other hand, GSH levels were significantly increased in

NAC group in comparison with the levels of MCT group

(GSH level 37.05� 3.94 mM in control and 32.85� 3.56 mM

in MCT, 44.71� 7.12 in NAC groups; Figure 4).

Liver MDA levels

MDA levels of liver tissue were observed to increase

with MCT application in comparison with control group.

MDA levels of NAC group were decreased when compared

with the levels of MCT group (MDA level 56.07� 7.85 mM

in control, 106.77� 5.66 mM in MCT, 52.04� 8.86 in NAC

groups; Figure 5).

Figure 4. The mean GSH levels of liver tissues in groups. C, controlrats (n¼ 10); MCT, monocrotaline-treated rats for 21 d (n¼ 10); NAC,N-acetyl cysteine: after 2 h from MCT injection. NAC (100 mgkg�1)was administered intraperitoneally for 21 d (n¼ 10). *Different from C(p50.05).

Figure 1. Assessment of right ventricular pressure in anesthetizedrats. C, control rats (n¼ 10); MCT, monocrotaline-treated rats for21 d (n¼ 10); NAC, N-acetyl cysteine: after 2 h from MCT injection.NAC (100 mgkg�1) was administered intraperitoneally for 21 d (n¼ 10).*Different from C (p50.05); ***different from C (p50.01);þþþdifferent from MCT (p50.01).

Figure 2. Assessment of right ventricular hypertrophy. C, controlrats (n¼ 10); MCT, monocrotaline-treated rats for 21 d (n¼ 10); NAC,N-acetyl cysteine: after 2 h from MCT injection. NAC (100 mgkg�1)was administered intraperitoneally for 21 d (n¼ 10). *Different from C(p50.05).

Figure 3. The mean AST levels of liver tissues in groups. C, controlrats (n¼ 10); MCT, monocrotaline-treated rats for 21 d (n¼ 10); NAC,N-acetyl cysteine: after 2 h from MCT injection. NAC (100 mgkg�1)was administered intraperitoneally for 21 d (n¼ 10). *Different from C(p50.05).

Table 1. The mean ALT, GGT and AP levels of liver tissues in groups.

C MCT NAC

ALT (IU L�1) 77.15� 12.30 79.54� 24.53 77.43� 25.49AP (IU L�1) 574.87� 189.63 564.22� 268.97 602.24� 320.97GGT (IU L�1) 5.14� 2.77 5.56� 3.40 5.26� 3.07

No significant difference was observed between groups. C, controlrats (n¼ 10); MCT, monocrotaline-treated rats for 21 d (n¼ 10); NAC,N-acetyl cysteine: after 2 h from MCT injection. NAC (100 mgkg�1)was administered intraperitoneally for 21 d (n¼ 10).

DOI: 10.3109/15569543.2013.809547 Protective effects of N-acetyl cysteine on MCT toxicity 49

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Light microscopic analysis of the pulmonary artery

In the liver tissue of the control group, normal vena centralis

structures and hepatocyte cells were observed. In the liver

tissues of the MCT group, necrotic areas were observed,

especially in hepatocyte cells. In the NAC group, normal liver

structures were observed (Figure 6).

Discussion

MCT phytotoxin, a pyrrolizidine alkaloid, has well-docu-

mented hepatic and cardiopulmonary toxicity for animals and

humans. Cratolaria species, which contain MCT, have been

widely used as a vegetable for the preparation of herbal teas or

medicines in Africa and India. They are used as colic, for

hemoptysis, against skin diseases topically and also are

taken for the relief of fever (IARC, 1972). A major exposure

of humans to MCT and related alkaloids was reported in West

India because of the consumption of extracts of Crotalaria

species as bush teas and an educational campaign was

developed to stop the consumption of Crotalaria teas in 1959,

which significantly reduced the incidence of veno-occlusive

disease, the major effect of the alkaloid (IARC, 1972;

Klaassen, 2001).

MCT increases pulmonary artery pressure and induces

pulmonary vascular remodeling with developing pulmonary

hypertension (Kodama & Adachi, 1999; Schermuly et al.,

2004). It causes pulmonary artery neomuscularisation, hyper-

plasia and hypertrophy of smooth muscle cells, inflammation,

and endothelial damage as well as the obliteration of

pulmonary vasculature and resultant right ventricular hyper-

trophy and failure (Latcham, 2005; Tuder et al., 1998).

Pulmonary vascular damage on endothelium which is induced

by MCT has a critical importance (Mathew et al., 1997;

Wanstall & O’Donnell, 1992). Interstitial edema, inflamma-

tion, hemorrhage and fibrosis are also observed defects in

liver with MCT (Baybutt et al., 2002; Yamashita et al., 2002;

Yee et al., 2000). Two phases has been observed, acute and

chronic, in hepatic MCT toxicity. Sinusoidal endothelial cells,

central venular endothelial cells and hepatic parenchymal

cells are damaged in early acute phase. After early lesions,

fibrotic occlusion, veno-occlusive syndrome or sinusoidal

obstruction syndrome developed in sub-lobular and sinusoidal

zones (Copple et al., 2002a, 2006).

In our study, RV pressures, which were increased signifi-

cantly in MCT group when compared with control group,

demonstrated compatibility with an increase in RV pressures

in groups with pulmonary hypertension in similar studies

(Clozel et al., 2006; Itoh et al., 2003; Kodama & Adachi,

1999). It is well known that mediators playing an important

role in the regulation of vascular tone are released from the

endothelium. The constant variations in the production of

vascular cell mediators cause impairment of homeostatic

balance; consequently, they effect vascular tone and induce

vascular remodeling (Levine, 2006; Toher, 2005). Because

of the endothelial damage induced by MCT, an increase is

expected in RV pressure depending on the imbalanced

homeostasis in the pulmonary vascular system. In NAC

group, RV pressures were reduced significantly when

compared with the values of MCT group. This finding was

demonstrated that this agent might have a protective effect

on MCT-induced pulmonary toxicity. Studies showed that

hepatic GSH levels were decreased depending on MCT

application (Wang et al., 2000). It is known that NAC is a

precursor of GSH and it exhibits protective effects with

a reduce in GSH levels or oxidative stress in diseases such

as cancer and heart diseases (Kelly, 1998; Raza et al., 2003;

Soto-Blanco et al., 2001; Tariq et al., 1999). NAC, which

is used to scavenge electrophilic metabolites of AAP, might

have also prevented the excessive formation of MCT pyrrole

and by this way reduced the amount of the reactive metabolite

reaching pulmonary tissue. On the other hand, various

antioxidants are also shown to have beneficial roles in

pulmonary hypertension. They can prevent the cellular

infiltration by altering the increased vascular permeability

under hypoxia (Devadasu et al., 2012). In a study, NAC

treatment during the initial stages of hypoxia prevented

pulmonary hypertension (Lachmanova et al., 2005). In our

study, the protective effect of NAC was shown in the dose

of 100 mgkg�1 in rats after administration of MCT with

reduced RVP. Pressure values in NAC group, which were

measured higher than those of the control group, indicated

that pulmonary hypertension was induced by MCT in our

study. In this case, 100 mgkg�1 of NAC might not be enough

for a complete protective effect for the increased RV pressures

in this model. In future, a study may be performed by higher

doses of NAC in steady pulmonary hypertension induced

with MCT.

Right ventricular hypertrophy, occurring in the late stages

of pulmonary hypertension as an adaptive response to the

increased pulmonary vascular resistance and endothelial

damage, was determined to be due to the increased RV/HW

ratios in untreated MCT group when compared with the

values in the control group. MCT-induced right ventricular

hypertrophy was also demonstrated in other studies (Clozel

et al., 2006; Kodama & Adachi, 1999; Wang et al., 2011).

In NAC group, right ventricular hypertrophy was significantly

decreased in comparison with that in MCT group. As a

GSH precursor, NAC showed a protective efficacy with its

antioxidant effect against the development of right ventricular

hypertrophy by reducing RVP when compared with MCT

group. In previous studies, many of the antioxidants showed

Figure 5. The mean MDA levels of liver tissues in groups. C, controlrats (n¼ 10); MCT, monocrotaline-treated rats for 21 d (n¼ 10); NAC,N-acetyl cysteine: after 2 h from MCT injection. NAC (100 mgkg�1)was administered intraperitoneally for 21 d (n¼ 10). *Different from C(p50.05).


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protective effects on right ventricular hypertrophy (Devadasu

et al., 2012; Lachmanova et al., 2005). The protective effect

of NAC was shown in the initial phase of pulmonary

hypertension when the participation of free radicals in the

pathogenesis is the most. The mechanism of the protective

role of NAC is thought to be arising from the inhibition of

reactive oxygen species (ROS) and peroxynitirite (product

of superoxide and nitric oxide interaction). These substances

are potent activators of collagenases that play the major

role in collagen breakdown. This process and its products may

stimulate fibroproduction and smooth muscle proliferation

in the walls of peripheral pulmonary arteries. Thus, radical

injury initiates the remodeling of pre-alveolar vessels by the

changes of matrix proteins and NAC inhibits this effect

(Lachmanova et al., 2005).

Levels of GSH in hepatic tissue were decreased in MCT

group when compared with the levels of the control group, but

this difference was not statistically significant. It was shown

that the levels of GSH in NAC group were statistically higher

than the levels of the MCT group (Figure 3). In other studies,

it was determined that MCT caused a reduction in the stores

of GSH in hepatic sinusoidal endothelial stems and hepato-

cytes. It was shown that long-term infusion of GSH

suppressed the damage which was induced by MCT in liver

(Deleve et al., 1996; Levine, 2006). It was also observed that

MCT pyrrols which was the active metabolite of MCT caused

a reduction in GSH stores in liver. MCT pyrrols, which are

potent alkylating agents, were connected to the cellular DNA

and proteins immediately. MCT pyrrols are detoxified with

GSH conjugation (Devadasu et al., 2012; Wang et al., 2000;

Figure 6. Assessment of liver light microscopy analysis. C, control rats; MCT, monocrotaline-treated rats for 21 d; NAC, N-acetyl cysteine: after 2 hfrom MCT injection. NAC (100 mgkg�1) was administered intraperitoneally for 21 d. !, hepatocyte damage; *, normal vena centralis structures.


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Wanstall & O’Donnell, 1992). In a study of Maioli et al., it

was suggested that depletion of GSH stores were played a role

in cytotoxicity in hepatocytes which was induced by MCT.

In another study, GSH levels and GST activity were decreased

in hepatic tissue with MCT (Lachmanova et al., 2005). In our

study, NAC, which plays a major role in GSH depletion, was

thought to increase the number of GSH stores.

MDA levels of liver tissues were increased in MCT group

in comparison with the levels of the control group. In other

studies, it was shown that MDA levels were increased

with MCT application in serum and lung tissue samples,

but despite its known hepatotoxicity, there was less evidence

about liver MDA levels (Jin et al., 2008; Wang et al., 2011).

Lipid peroxidation is a well-established mechanism of cellular

injury and it is used as an indicator of oxidative stress in

cells and tissues. Polyunsaturated fatty acid peroxides gener-

ate MDA and 4-hydroxyalkenals upon decomposition.

Measurement of MDA and 4-hydroxyalkenals is used as an

indicator of lipid peroxidation (Janero, 1990; Requena et al.,

1996). In our study, MCT induced lipid peroxidation in liver

and thereby free radical formation. NAC inhibited this effect

because of its antioxidant properties against different types

of toxicities as shown in the previous studies (Ronisa et al.,

2005; Yedjou et al., 2008).

In addition to the pulmonary toxicity of MCT, hepatotoxic

effects were also determined through the increases in serum

AST levels in MCT group when compared with the levels of

the control group. However, ALT, GGT and ALP levels,

which were the other markers of liver toxicity, were not

increased significantly in MCT group. In other studies, it was

shown that AST was present in tissues, such as heart, skeletal

muscle, kidney, brain and increases during heart attack,

whereas ALT is primarily localized in liver and increases

during any hepatic damage thereby is called the clinical

chemistry gold standard of hepatotoxicity (Ozer et al., 2008;

Srivastava & Chosdol, 2007; Thapa & Walia, 2007).

In conclusion, dose of MCT administered may be increased

to obtain significant hepatotoxicity. Levels of AST, which

were increased in our model, may be a predictor of MCT-

induced cardiotoxicity as well as other related toxicities or

the early stages of the hepatotoxicity induced by MCT.

In previous studies, injection of MCT in rats produced

dose-dependent hepatic parenchymal cell injury which was

significant at 200 mgkg�1. Injection of 300 mgkg�1 MCT

produced time-dependent hepatotoxicity with significant

injury beginning by 12 h after treatment (Copple et al.,

2002b, 2003). Doses higher than 100 mgkg�1 of MCT may be

preferred to develop severe hepatotoxicity in rats (Joseph

et al., 2006; Yee et al., 2002). In histological sections, a more

marked vena centralis and hepatocyte deteriorations in liver

structure were observed in the MCT group in comparison with

the control group. This finding demonstrated that MCT had

effects of injury on structural parameters of liver in our study.

Therefore, even hepatic damage was initiated by MCT, it was

not an appropriate model to research the hepatoprotective

effect of an agent, at the dose of 60 mgkg�1 MCT. In addition,

protective effects of NAC were identified with the decrease

of serum AST level and with amelioration in hepatic tissue

histopathologically in our study. In conclusion, NAC was

thought to be hepatoprotective against the initial hepatotoxic

signs of MCT in our model. Also, in a study of Yamashita

et al., the hepatic damage, which was induced by MCT,

was presented with elevated serum AST and ALT levels.

The protective role of exogenous GSH administration was

determined in MCT-induced hepatic damage. It was sug-

gested that GSH stores in sinusoidal endothelial cells were

related with detoxification of reactive MCT pyrrole metab-

olites and GSH conjugation had an important role in MCT-

induced hepatotoxicity model (dos Santos et al., 2009; Maioli

et al., 2011). The hepatoprotective effects of NAC were

shown in AAP-induced hepatotoxicity models. In these

studies, serum ALT and AST levels were decreased in NAC

group when compared with the levels in the AAP group.

It was shown that ROS scavenger effects of cysteine analogs

contributed to this protective activity (Acharya & Lau-Cam,

2010; Terneus et al., 2008). In another study, the protective

effect of NAC was shown against DNA damage and hepatic

toxicity induced with radiation. This condition was attributed

as a result of the increase in radiation-dependent reduced

GSH levels in liver after NAC administration (Mansour et al.,

2008). In a study of Raza et al., NAC showed protective

effects by increasing GSH stores against hepatic damage

induced with azathioprine. This condition was demonstrated

that NAC provided the substrate of sulfhydryl group for

hydroxylation of depleting GSH stores and other free radicals.

According to our results, it was observed that pulmonary

toxicity was occurred with MCT injection and the severity of

the toxicity was suppressed by NAC administration protect-

ively. This toxic effect was related to MCT and was depended

on the electrophilic MCT pyrrole metabolites which were

formed by hepatic cytochrome oxidases. This electrophile

groups damage DNA and proteins in cells. Hepatic and

pulmonary GSH stores also play an important role in

detoxification of these electrophilic metabolites. Lack of

GSH stores and/or increases in metabolite formation result in

vulnerable cells and also cause cellular damage (Amin et al.,

2011; Deleve et al., 1996). NAC is a source of L-cysteine for

GSH and it could increase the detoxification of MCT

metabolite which induced hepatic and pulmonary toxicity.

In our study, NAC, as an antioxidant, is thought to have shown

preventive effect against oxidative stress in pulmonary

vascular system with increasing GSH stores.

In our study, pulmonary toxicity was shown by the increase

in the RVPs and right ventricular/heart ratios induced by

MCT. However, despite an increase in serum AST levels,

similar values of the ALT, GGT and AF levels between

control group and MCT group, indicated that hepatotoxicity

was initiated by MCT but hepatic damage was induced

inadequately at the dose of 60 mg kg�1. NAC administration

showed a protective effect in MCT-induced pulmonary

hypertension model as a result of the significant reduction

in RV pressures and also in RV/HW ratios when compared

with the values of the MCT group. NAC also showed its

protectivity in the early stages of hepatotoxicity in this model

by the reduction in the levels of biomarkers of hepatic damage

and also by the recovery in histopathological examination.

Consequently, although pulmonary toxicity was induced at

the dose of 60 mgkg�1 intraperitoneally by MCT application,

hepatotoxicity was not observed completely. It may be

suggested that the protective and therapeutical effects of the


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agents can be investigated by inducing hepatotoxicity with

MCT application in higher doses.

Conclusion

In this study, pulmonary toxicity and hepatotoxicity of MCT

were investigated while the toxicity of MCT on different

organs or systems was investigated in previous studies

separately. Thereby, our study may represent a well-arranged

report of pulmonary toxicity and hepatotoxicity of the

phytotoxin, MCT. NAC may be a protective agent against

pulmonary toxicity and hepatoxicity of MCT by the results

of RVP, RV hypertrophy, hepatic and serum enzyme levels

measured and histopathological examinations. For determin-

ing this effect in details, hepatotoxicity may be induced by

higher doses of MCT. In future, the effect of NAC in early

stages of PH can be tested because of the similar toxicity

mechanisms of MCT and AAP. In this aspect, the aim is

to investigate the reversibility of the toxic effect by NAC.

In vascular wall, the metabolization pathway of MCT has not

been tested before. According to our results, the amelioration

in the parameters of pulmonary toxicity by NAC indicates the

necessity of illuminating the metabolization process of MCT

in vascular wall and pulmonary tissue.

Declaration of interest

The authors report no conflicts of interest. The authors alone

are responsible for the content and writing of this article.

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is n -acetyl cysteine protective against monocrotaline-induced toxicity?

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